The overarching theme of the proposed research plan is to advance our ability to engineer natural product biosynthetic pathways for the production of candidate drug-like molecules. Natural products produced in Nature display a broad range of chemical functionalities, shapes, and sizes, imbuing many of these molecules with important therapeutic benefits that include anticancer and antibiotic activities. By increasing our understanding of these biosynthetic pathways at the atomic level, we will be able to manipulate different natural product systems to produce target compounds of chemical interest for drug discovery. This proposal aims to characterize the molecular and biochemical features of five-membered heterocycle formation in nonribosomal peptide synthetases (NRPSs), a family of natural products responsible for the generation of peptides and peptide/polyketide hybrid molecules such as the anticancer agents bleomycin and the epothilones, as well as the antibiotic bacitracin. The NRPS heterocyclization (HC or Cy) domain is responsible for installing five-membered thiazoline or oxazoline rings within NRPS products, originating from the amino acids cysteine or serine/threonine, respectively. The presence of these heterocycles increases compound stability and modifies the 3D shape of the natural products, therefore they play extremely important roles in defining biological activity and the ability to engineer these rings into novel compounds is of utmost interest. Aim 1 entails crystallizing different designed HC domain constructs in order to obtain a series of X-ray crystal structures of different HC domains from the epothilone and yersiniabactin biosynthetic gene clusters. These results will provide a direct comparison of how different HC domains from a related system are modified to accommodate differently sized substrate molecules. The second aim of this proposal focuses on rationally modifying substrate-binding residues within the HC domain and characterizing the resulting changes on substrate preferences and reaction kinetics. This aim will translate information regarding these structures to engineering applications, an important step to guide engineering of this system for future drug design. The overall strength of this AREA proposal is in the integrated approach at defining the activity of the NRPS HC domain for bioengineering efforts. This work combines the use of protein construct design, X-ray crystallography, mutagenesis, and biochemical activity assays to characterize the molecular function of the HC domain. In addition to directly involving undergraduate and graduate students in research, these results will impact multiple fields that include natural product biosynthesis, bioengineering, protein design and biocatalysis.